The investigator indicates that the only well-characterized HIV-1 neutralizing antibodies to date are directed against a hypervariable region of the gp120 glycoprotein. HIV-1-infected individuals, however, generate antibodies capable of neutralizing diverse HIV-1 isolates, suggesting that the epitopes are conserved among HIV-1 variants. The mixed nature of patient antibody responses and the fact that many of these important epitopes are discontinuous have made characterization difficult. The goal of this proposal is to characterize, at the molecular level, the HIV-1 gp120 epitopes and the antigen combining sites of monoclonal antibodies that exhibit broad neutralizing activity. Discontinuous gp120 epitopes will be mapped by testing a large panel of gp120 mutants for ability to be recognized by individual monoclonal antibodies. Potential neutralization escape nutants will be identified. The gp120-combining site of selected monoclonal antibodies will be characterized at the structural level. These studies will serve as a basis for expression of active antibody fragments in E. coli. Mutant antibody fragments will be generated and screened for altered affinity or specificity for the HIV-1 gp120 glycoprotein.